Field of the Invention
The present invention relates to devices for processing biological samples, especially for amplifying DNA sequences by the Polymerase Chain Reaction (abbreviated “PCR”) method. In particular, the invention concerns a thermal cycler comprising a sample holder portion as well as a microtiter plate designed to be placed in a holder of a cycler device.
Description of Related Art
Thermal cyclers are instruments commonly used in molecular biology for applications such as PCR and cycle sequencing, and a wide range of instruments are commercially available. A subset of these instruments, which include built-in capabilities for optical detection of the amplification of DNA, are referred to as “real-time” instruments. Although these can sometimes be used for different applications than non-real-time thermal cyclers, they operate under the same thermal and sample preparation parameters.
The important parameters that govern how well a thermal cycler operates are: uniformity, accuracy and repeatability of thermal control for all the samples processed, ability to operate in the environment of choice, speed of operation, and sample throughput.
The uniformity, accuracy and repeatability of thermal control is critical, because the better the cycler is in these parameters, the more confidence can be placed in the results of the tests run. There is no threshold beyond which further improvement in these parameters is irrelevant. Further improvement is always beneficial.
The ability to operate in the environment of choice is not a problem for devices used in a laboratory setting where the samples are brought to it, but choices become limited when it is desired to use the instruments outside the laboratory and to bring it to where the samples are located. The two main concerns here involve the size and, thus, portability of the instrument, and the power requirements of the instrument. These two concerns are directly related, as the biggest single component in most cyclers is the heat sink used to reject the waste heat generated by the cycling. If a thermal cycler were to be built such that it only required enough power to operate off an automobile battery, it would also use a smaller heatsink because less waste heat was being generated and it would become portable enough to operate virtually anywhere on earth.
Thermal cycling speed is important not just because it is a major factor in determining sample throughput, but also because the ability to amplify some products cleanly and precisely is enhanced or even enabled by faster thermal ramp rates. This can be particularly true during the annealing step that occurs on each cycle of an amplification protocol. During that time, primers are bonded onto the templates present, but if the temperature is not at the ideal temperature for this, non-specific bonding can occur which in turn can lead to noise in the results of the reaction. By increasing ramp rate, the time that the reaction spends at non-ideal temperatures is reduced.
Sample throughput needs have come about over time. All currently produced thermal cyclers can be divided up into groupings based on how they accommodate samples. The first instruments were built to accommodate a small number of tubes which were individually processed and loaded into the cycler (example: Perkin-Elmer 4800). As sample throughput needs grew, instruments were developed to accommodate plastic trays (microtiter plates) that were essentially arrays of 96 or 384 tubes (examples: Perkin-Elmer 9600, MJ Research PTC-200, Eppendorf MasterCycler). Both of these approaches utilized metal blocks to heat and cool the tubes, which places some limits on the speed of thermal cycling due to the time needed to heat and cool the mass of the metal. Another approach to increase sample throughput focused on decreasing the time needed to process a batch of samples by speeding up the rate of thermal transfer to the samples, and these systems utilized glass capillaries or proprietary sample holders (examples: Idaho Technologies RapidCycler, Cephied Smartcycler, Analytik Jena Speedcycler). The last category of thermal cycler instruments are built around microfluidics-based sample holders, but have not been widely used due to the limited fluid volume of the samples they can process and the difficulty of preparing the microfluidics sample holders.
The vast majority of thermal cyclers in use today are in the second grouping: block based thermal cyclers that accommodate microtiter plates. The reason for this, despite the lower cycling speed of these instruments, is that microtiter plates can be used with a wide range of liquid volumes, and the actual sample throughput is higher in terms of total number of samples that can be processed in a given timeframe. This last is only partially a function of the instrument itself; it is also due to the equipment that is available to process and load the samples. The vast majority of microtiter plates in use conform to a set of standards codified by the Society for Biomolecular Screening (SBS) over the last decade. The plates typically have 6, 24, 96, 384 or even 1536 sample wells arranged in a 2:3 rectangular matrix. The standard governs also well dimensions (e.g. diameter, spacing and depth) as well as plate properties (e.g. dimensions and rigidity).
A number of robots designed to specifically handle SBS microplates have been developed. These robots may be liquid handlers which aspirate or dispense liquid samples from and to these plates, or “plate movers” which transport them between instruments. Also plate readers have been developed, which can detect specific biological, chemical or physical events in samples being processed in the plates.
Adherence to the SBS Microtiter Plate Standards, has allowed for easy integration of robotics solutions, such as liquid handling machines, into the sample preparation process, which has had a profound impact on the ability to increase sample throughput.
It can therefore be concluded that technical solutions that will further increase sample throughput must do so without compromising the ability to work within the SBS specifications. Though having assisted with the spread of cycler instruments and harmonized the processes used by different device manufacturers, some specifications of SBS standards have also limited further development of cycler instruments by narrowing down the scope of research, for example, as far as power consumption and cycling rate are concerned.
In a set of laboratory protocol steps, in which PCR is one of those steps, PCR is often the rate limiting step. Thus, a primary objective of those familiar with the process is to decrease the overall time required to perform PCR.
Some known thermal cyclers having traditional heat transfer components are presented in the WO-publications 03/061832 and 2004/018105 and in the GB-publication 2370112.